PROJECT SUMMARY This is the competitive renewal of our program on MITF, a bHLHzip transcription factor that we have shown to play a master regulatory role in melanocytes. Our accomplishments include the discovery that MITF transcriptionally regulates nearly all known melanocyte-specific mediators of melanin biosynthesis. We linked MITF expression to Melanocyte Stimulating Hormone/MC1R signaling and to post-translational modulation by c-Kit/MAPK, and utilized these insights to molecularly define the UV-tanning pathway. We generated and analyzed red hair/light skin models of carcinogenesis, and discovered an endorphin-mediated behavioral impact of UV irradiation to skin. Our studies identified MITF target genes and biological behaviors that impact skin cancer prevention (through potent pigmentation effects) and oncogenesis (if genomically mutated). In the most recent grant period, we generated abundant data, including discoveries that set the background for the current application. In profiling MITF target genes, we unexpectedly observed MITF occupancy and regulation of the PDL1 gene. This odd connection between MITF and a key ubiquitous immune-tolerance factor was also notable because vitiligo patients are known to exhibit significantly lower-than-expected non-melanoma skin cancer incidence. This led us to consider that PDL1 may exert tolerance for accumulation of UV-induced high- neoantigen burdens, particularly in non-regenerating cells. Beyond extensive molecular analyses, we observed vitiligo-like melanocyte depletion with immune infiltration in UV-irradiated PDL1-deficient mice. This suggests an opportunity to use PD1 suppression for immune clearance of neoantigen-accumulating skin cancer precursors in high-risk individuals?which we will test in preclinical models (Aim 1). We have deeply analyzed red hair/light skin phenotypes, as well as UV-dependent and independent melanoma risk. In mouse models, we observed several cAMP-inducing agents to rescue dark/eumelanin pigmentation and protect against UV carcinogenesis. However, these agents could not topically penetrate human skin sufficiently to affect pigmentation. We now study an alternative target, SIK, whose kinase suppression induces CREB and MITF, as well as pigmentation. Systemic SIK inhibitors are being studied in a variety of diseases, and we found topical administration induces strong eumelanin-darkening in both human and mouse skin. Given potential translation to humans, we will evaluate this for skin cancer prevention using preclinical models (Aim 2), and test its effects for safety on BRAF- or NRAS-induced nevi in mice. Finally, we discovered MITF as a transcriptional repressor of numerous genes involved in neuron or Schwann cell development. Some of these are known to, themselves, antagonize MITF expression. This suggests a mutually repressive developmental ?double-switch? to determine lineage destiny for neural crest derivatives. We will (Aim 3) scrutinize mechanisms underlying MITF's repressive activity, use models to biologically test such lineage switching, and test repressed targets for expression in a low-MITF melanoma subtype (desmoplastic) that is in need of diagnostic markers.